Molecular Biochemical Study on the Cyanobacterial ndh (NADH dehydrogenase) Gene Products

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Eleven ndh genes (ndhA-K) have been identified in chloroplast and cyanobacterial genomes on the basis of sequence similarity to the subunits of mitochondrial and bacterial NADH : ubiquinone oxidoreductase. These findings have been taken to indicate the presence of an enzyme related to the mitochondrial and bacterial enzymes, possibly an NAD (P) H : plastoquinone oxidoreductase ; however, the active enzyme complex has not yet been isolated. In order to clarify its physiological significance, several attempts have been made using the filamentous cyanobacterium Plectonema boryanum, a facultative chemoheterotroph.Three essential ndh genes are missing from the plastid genomes. They are 51 (FP), 24 (FP), and 75 (IP) subunits of bovine complex I,which constitute a subcomplex involved in the first part of electron pathway from NADH to intermediate acceptors. Cyanobacterial DNA fragment was cloned after PCR amplification, which shows sequence similarity to the mitochondrial 51 (FP) subunit. Attempts to inactivate the ndhA,ndhI,ndhG,or ndhE gene of P.boryanum have been unsuccessful under both normal and high-CO2 growth conditions, suggesting an essential role in this organism. In order to identify ndh related genes, we have developed a system for mutagenesis using a non-replicating plasmid pTAF10 which was constructed from pUC12, the neo gene, and OMEGA fragment. Genomic fragments partially digested by Sau3A were ligated into pTAF10, introduced into the cells by electroporation and kanamycin resistant mutants were obtained after single cross-over recombination. Insertion of the entire plasmid into the chromosome may interrupt the gene or disturb the expression of flanking genes due to the terminator sequence in the OMEGA fragment. Screening for the defect in chemoheterotrophic growth resulted in the isolation of dozens of mutants which are now analyzed.